Nucleic acid treatment is a therapeutic method for suppressing expression of a pathogenic protein by delivering the nucleic acid (RNA) into the cytoplasm, and gene therapy is a therapeutic method for promoting expression of a protein useful for the treatment by delivering the nucleic acid (DNA) into the nucleus. In these treatment methods, delivery of the nucleic acid into cells is important. However, delivery of nucleic acid into cells is difficult, since nucleic acid is rapidly degraded when used alone by enzymes in the blood. Therefore, practicalization of these therapeutic drugs requires a carrier for delivery of the nucleic acid into the cells.
In view of the property of the carrier that delivers foreign substances into cells, it is necessary to exhibit a large effect with a small amount of use. That is, the nucleic acid delivery carrier is required to increase the delivery amount of the nucleic acid per unit carrier incorporated into the cytoplasm, that is, to increase the nucleic acid delivery efficiency into the cytoplasm.
Virus vectors represented by retrovirus and adenovirus are carriers with high nucleic acid delivery efficiency. On the other hand, they are associated with problems such as formation of tumor caused by insertion of the viral vector into the genome and nonspecific influence on cells other than the target cells. In view of these, the development of non-viral carriers is ongoing. Of those, a nucleic acid delivery carrier using a cationic lipid (lipid membrane structure) is a non-viral carrier used most generally.
To increase nucleic acid delivery efficiency with a nucleic acid delivery carrier using cationic lipid, pharmacokinetics (e.g., stability in blood, accumulation property in target cells such as tumor and the like, and the like) need to be improved. Furthermore, to increase delivery efficiency of nucleic acid into the cytoplasm, improvement of intracellular dynamics (e.g., uptake into cells, escape from endosome, release of nucleic acid from carrier in the cytoplasm and the like), besides the aforementioned pharmacokinetics, also becomes necessary (non-patent document 1).
Cationic lipids are roughly composed of a hydrophobic moiety and a hydrophilic moiety. As its constitution, the hydrophobic moiety comprises a hydrophobic group such as fatty acid group, sterol group and the like, and the hydrophilic moiety comprises a cationic group such as amino group, and the like. As the composition of the cationic lipid, many structures comprising two hydrophobic groups per one hydrophilic group (hereinafter to be referred to as “two-chain cationic lipid”) are known.
As mentioned above, a nucleic acid delivery carrier using a cationic lipid requires improvement of pharmacokinetics and intracellular dynamics. Since nucleic acid and cellular membrane are anionic, the cationic group of a cationic lipid, which electrostatically interacts with them, has been found to play an important role in solving these problems. Therefore, among cationic lipids, cationic groups, namely, amino groups, are being developed mainly.
For improvement of intracellular dynamics, a method using a cationic lipid having a quaternary amine is known. For example, known two-chain cationic lipid 1,2-Dioleoyl-3-dimethylammonium propane (hereinafter to be referred to as “DOTAP”) having a quaternary amine can form a positively-charged lipid membrane structure by an electrostatic interaction between an amino group of DOTAP and an anionic nucleic acid. The positively-charged lipid membrane structure interacts with an anionic cellular membrane to increase uptake into the cell. However, since the electrostatic interaction between DOTAP having a quaternary amine and nucleic acid is too strong, release of the nucleic acid from the carrier is problematically difficult (non-patent document 2).
On the other hand, various studies have also been made on tertiary amine. As a known two-chain cationic lipid having tertiary amine, 1,2-Dioleoyl-3-dimethylamino propane (hereinafter to be referred to as “DODAP”) can be mentioned. It is described that DODAP can form a lipid membrane structure by electrostatic interaction with nucleic acid, and becomes a carrier capable of delivering nucleic acid to the target cell (non-patent document 2).
Non-patent document 3 describes pharmacokinetics. In this document, pKa of two-chain cationic lipid is adjusted to near neutral. It is shown that a lipid membrane structure using the cationic lipid is stable in blood for a long time after intravenous injection, and accumulated in the tumor site.
Non-patent document 4 describes intracellular dynamics. This document describes as regards two-chain type cationic lipids that pKa as a lipid membrane structure can be adjusted to a value advantageous for intracellular endosomes escape, by changing the structure around the amino group. It is stated that this promotes endosomal escape and clearly improves nucleic acid delivery efficiency.
In addition, cationic lipids having hydrophobic group and tertiary amino groups with different amino group number have also been developed. For example, patent documents 1 and 3 describe cationic lipids having a structure in which compounds having one hydrophobic group and one hydrophilic group are linked with each other by a biodegradable disulfide bond. The documents show that the cationic lipid can improve pharmacokinetics such as stability in blood, tumor targeting property and the like. In addition, it has been clarified that the cationic lipid can improve intracellular dynamics such as increase in the delivery efficiency of nucleic acid into the cytoplasm and the like, since it exhibits higher nucleic acid delivery efficiency as compared to known cationic lipids such as DOTAP and DODAP.
However, despite the technical progress in this field, the nucleic acid delivery efficiency into the cytoplasm, which is achieved by a lipid membrane structure using cationic lipid, is not fully satisfactory.
As described in patent document 2, it is useful to contain many amino groups when intracellular deliverability is to be improved. However, when many amino groups are contained, release of nucleic acid from the carrier in the cell is suppressed. Therefore, improvement of nucleic acid delivery efficiency cannot be expected.